PROJECT SUMMARY Genome instability caused by incorrect DNA repair system is a major driver for tumorigenesis. Our long- term goal is to understand how cellular proteolysis controls the pathway responsible for repairing DNA damage, thereby preserving the integrity of the genome. Since homeostasis of DNA repair factors is critical for the activity of DNA repair, elucidating underlying mechanisms for the ubiquitin-proteolytic pathway in DNA repair is essential for understanding the etiology of cancer when it is derailed. We are interested in the mechanisms that link proteolysis to signaling of the Fanconi anemia (FA) DNA repair pathway, which deals with DNA interstrand cross-links (ICL) encountered during DNA replication. Its defects lead to a high risk of multiple cancers due to elevated genome instability, and its aberrant activity is known to influence therapeutic response to cytotoxic chemotherapy that utilizes DNA cross-linking agents including platinum. Thus, knowledge on molecular and genetic factors that control the FA pathway is expected to help us exploit their deregulation for the development of improved cancer therapeutics. One of the fundamental regulatory mechanisms for protein degradation is reversible phosphorylation of protein targets, which marks a protein to be destroyed by ubiquitin-proteasome system. We recently discovered the proteolytic signaling pathway of FAAP20, a key component of the FA core ubiquitin E3 ligase complex necessary for the FA pathway activation, and showed that deregulation of FAAP20 leads to a functional disruption of the FA core complex, impairing the ability of cells to repair DNA ICL lesions. Specifically, we defined SCFFBW7 as an ubiquitin E3 ligase complex responsible for phosphorylation-dependent FAAP20 degradation and demonstrated how its deregulation affects the FA pathway. Our preliminary studies also indicate that phosphorylation-dependent conformational change of FAAP20 regulated by cis-trans isomerase PIN1 modulates ubiquitin signaling of FAAP20 degradation, thereby determining the fate of the FA core complex and influencing the efficiency of DNA ICL repair. Herein, we propose to explicate PIN1-SCFFBW7 proteolytic signaling in controlling the FA pathway and its impact to genome instability. Specifically, we will (1) dissect the signaling pathway of FAAP20 degradation regulated by SCFFBW7, (2) elucidate the mechanisms by which PIN1-driven structural change of FAAP20 functions as a regulatory switch to control FAAP20 stability, and (3) determine the role of PIN1 in regulating DNA ICL repair and the therapeutic response of breast cancer to platinum via FA pathway signaling using cancer cell lines and a mouse model. Together, our studies are expected to reveal the first direct link between a highly deregulated PIN1-SCFFBW7 axis in human cancer and DNA ICL repair. This work will ultimately benefit human health by offering a unique opportunity to design therapeutic interventions that exploit aberrant DNA repair-associated proteolytic signaling in FA-related malignancy and cancer in general.